Flaviviruses are a group of positive-stranded RNA viruses that have a global impact due to their widespread distribution and ability to cause disease in humans and economically important domesticated animals. Several members of this genus, such as dengue virus (DENV) and West Nile virus (WNV), are considered emerging or re-emerging pathogens because the incidence with which they encounter humans and cause disease is increasing each year at an alarming rate. There are more than 50 million humans infected by flaviviruses each year. WNV is a mosquito-borne member of this genus and is the etiologic agent of West Nile encephalitis. WNV is endemic in parts of Africa, Australia, Europe, Asia, and the Middle East and has been responsible for periodic outbreaks of encephalitis in humans and horses. The introduction of WNV into North America in 1999 and its rapid spread across the United States into Canada, Mexico, and the Caribbean identifies this virus as an emerging pathogen of clinical and economic significance for the Western Hemisphere. While seroprevalence studies indicate that most WNV infections of humans are subclinical, clinically apparent infections range from a febrile illness (West Nile fever) to more severe and potentially fatal neurologic disease. Currently, no WNV vaccine has been approved for use in humans and treatment is supportive. Cryo-electron microscopy studies reveal that the surface of flavivirus virions is covered by a highly ordered icosahedron composed of 180 envelope proteins. The generation of antibodies capable of binding to this array of viral proteins and blocking infection is a critical aspect of the immune response and an important goal of vaccine development. Passive immunization studies and experimental infections of immunodeficient mice demonstrate that antibody plays a significant role in protection from flavivirus infection. The importance of antibodies in vivo reflects an ability to directly neutralize virus infectivity, as well as several distinct effector functions mediated by constant portions of the antibody molecule. The neutralization potential of an antibody is governed by the number of sites on the virion available for binding (determined by epitope accessibility) and the strength of binding (antibody affinity/avidity). Using neutralizing antibodies that bind structurally distinct sites on the WNV or DENV virion, we are investigating the biochemical basis of potency with respect to how antibodies engage virus particles, and in what numbers. Our recent data suggest that neutralization of WNV requires engagement of individual virions with a stoichiometry that exceeds a required threshold. Our estimate for this threshold is roughly 30 antibodies. Due to the quasi-icosahedral symmetry of the mature virion, not all epitopes on the flavivirus are equally accessible for antibody binding. Accessibility modulates antibody potency, as some determinants may not be exposed with a frequency that permits neutralization. Thus, antibodies that recognize such epitopes may neutralize poorly, or not at all, even at concentrations that permit saturation because too few antibodies can simultaneously dock on the virion. Of interest, many antibodies that recognize poorly exposed epitopes on the mature virion still show neutralizing activity in vitro and protect in vivo. How antibodies engage cryptic epitopes on virions with a stoichiometry that permits neutralization is difficult to reconcile using existing static models of virion structure and envelope organization. To investigate mechanisms that govern the potency of antibodies that target cryptic epitopes, we are investigating the dynamics that control epitope accessibility and neutralization potency. Antibody-dependent enhancement of infection. Paradoxically, antibodies may also play a role in enhancing virus infection and exacerbating disease. Antibody-dependent enhancement of infection (ADE) describes a dramatic increase in infection of Fc-receptor-bearing cells in the presence of sub-neutralizing concentrations of antibody or immune sera. The most direct link between ADE and the clinical outcome of DENV infection comes from investigations of the unusually large number of DHF cases following primary infection observed in infants during the first year of life. At birth, DENV-specific passively acquired antibodies are present at a relatively high concentration and exhibit neutralizing activity in vitro. However, as the child ages, degradation of maternally acquired antibody continues to levels that are no longer protective, do not neutralize virus, and enhance virus infection of Fc-receptor-expressing cells in vitro. The waning antibody titers of infants to levels that support ADE in vitro parallels the risk of DHF following primary DENV infection during the first year of life. In a broader context, antibodies elicited by primary infection with one serotype of DENV may bind related viruses introduced during secondary infection with reduced avidity, resulting in engagement of the virion with a stoichiometry that does not permit virus neutralization but can support ADE. The development of an immune response that elicits protective levels of neutralizing antibodies against all four serotypes of virus present in the vaccine is a key factor for reducing the risk of ADE. To facilitate this goal, our laboratory is investigating the biochemical determinants that comprise the enhancing character of an antibody, and the cell biology that underlies the mechanism of ADE.